Description: (Applicant's Description) This component within the overall NCDDG application is intended to improve the fundamental understanding and the use of E. coli purine nucleoside phosphorylase for cancer treatment. We have previously characterized E. coli PNP as a very efficient method for eliciting bystander killing of human tumor cells. The approach differs fundamentally from the HSV-tk and other prodrug activation strategies since it a) provides a toxin that kills tumors with a low growth fraction, b) generates a toxin that diffuses between and among tumor cells with potent bystander activity (even when less than 1 in 100 cells express the gene) and c) does not require cell-to-cell contact or gap junctions in order to mediate these effects. Laboratory Program 1 will characterize boundaries of bystander killing by E. coli PNP and provide important new information regarding toxicity of the strategy. The experiments will also develop novel vectors for delivery of E. coli PNP to pre-establish tumors in mouse models. Finally, these studies are intended to examine recent information developed in this NCDDG about the active site of E. coli PNP, and ways the crystal structure of the enzyme can facilitate development of better prodrug/enzyme combinations.